In the past, research on RNA virus vectors has been carried out using infectious clones of the tobacco mosaic virus (TMV) and bromo mosaic virus (BMV), which have extremely high proliferative capability in infected plants. In this field several strategies for expressing foreign genes have been developed in the past because of differences in viral gene expression patterns, etc.
As an introduction to such strategies, for example, protein expression vectors prepared by fusion of a virus coat protein (CP) gene and a foreign gene have been developed using TMV and potyvirus (see non-patent documents 1 and 2). In addition, a vector expressing a foreign gene via a subgenome has been developed by transfection of a subgenomic promoter of TMV and a closely related virus (Odontoglossum ringspot virus) (see non-patent document 3).
However, rod-shaped and filamentous viruses typified by TMV and the family Potyviridae have primarily been used in the development of such virus vectors. That is because compared with spherical viruses, rod-shaped and filamentous virus vectors have the advantage of fewer physical limitations in the length of the foreign gene to be inserted. As an expression vector system using BMV, which is a spherical virus, a method has been reported wherein an RNA-3 derivative carrying a foreign gene was inoculated into tobacco expressing the replicase genes for protein 1a and protein 2a (see non-patent document 4).
Several studies using a cucumber mosaic virus (CMV) vector have also been conducted. In one such study a four-component CMV vector was developed by complementation (see non-patent document 5). It was found that both the movement protein (MP) and coat protein (CP) are encoded by RNA-3 of the CMV, and that both MP and CP are essential for intercellular movement of the virus (see non-patent document 6).
The inventors have already modified the RNA-3 molecule to construct an RNA-3A component wherein the MP region was replaced by the green fluorescent protein (GFP) gene and an RNA-3B component from which the CP gene was deleted, and thereby they have developed a virus vector capable of intercellular movement by complementation. When this vector was inoculated into Nicotiana benthamiana plants as a CMV having a virus genome consisting of the four components RNA-1, RNA-2, RNA-3A and RNA-3B, GFP fluorescence was observed in the minor veins of inoculated leaves.
In addition, when multiple cloning sites were inserted into the CP region that had been deleted in RNA-3B, and the GUS (bacterial-β-glucuronidase) gene and the bean yellow mosaic virus (BYMV) CP gene were inserted thereinto, GUS activity and the BYMV-CP were detected in the minor veins of inoculated leaves. However, homologous RNA recombination occurred between components RNA-3A and RNA-3B, and no systemic expression of the inserted genes was found.
In addition, as prior art various related techniques have been developed such as the production of various proteins by CMV vectors that can be used in a variety of different host plants (non-patent documents 7-11).    Non-patent document 1: Sugiyama, Y., Hamamoto, H., Takemoto, S., Watanabe, Y. and Okada, Y., Systemic production of foreign peptides on the particle surface of Tobacco mosaic virus, FEBS Letters 359, 247-250 (1995)    Non-patent Document 2: Fernandez-Fernandez, M. R., Martinez-Torrecuadrada, J. L., Casal, J. I. and Garcia, J. A., Development of an antigen presentation system based on plum pox potyvirus, FEBS Letters 427, 229-235 (1998)    Non-patent Document 3: Donson et al., Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector, Proc. Natl. Acad. Sci. USA 88:7204-7208 (1991)    Non-patent Document 4: Mori, M., Kaido, M., Okuno, T. and Furusawa, I., mRNA amplification system by viral replicase in transgenic plants, FEBS Letters 336, 171-174 (1993)    Non-patent Document 5: Zhao, Y., Hammond, J., Tousignant, M. E., and Hammond, R. W., Development and evaluation of a complementation-dependent gene delivery system based on Cucumber mosaic virus, Archives of Virology 145, 2285-2295 (2000)    Non-patent Document 6: Canto, T., Prior, D. A., Hellward K. H., Oparka, K. J., Palukaitis, P., Characterization of cucumber mosaic virus. IV. Movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus, Virology 237, 237-248 (1997)    Non-Patent Document 7: Matsuo, K., Hong, J. S., Tabayashi, N., Ito, A., Masuta, C., Matsumura, T. Development of Cucumber mosaic virus as a vector modifiable for different host species to produce therapeutic proteins. Planta 225:277-286 (2007)    Non-Patent Document 8: Baulcombe, D C., Chapman, S., Santa Cruz, S. Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7:1045-1053 (1995)    Non-Patent Document 9: Wang, Z. D., Ueda, S., Uyeda, I., Yagihashi, H., Sekiguchi, H., Tacahashi, Y., Sato, M., Ohya, K., Sugimoto, C., Matsumura, T. Positional effect of gene insertion on genetic stability of a clover yellow vein virus-based expression vector. J Gen Plant Pathol 69:327-334 (2003)    Non-Patent Document 10: Mitsuhara, I., Ugaki, M., Hirochika, H., Ohshima, M., Murakami, T., Gotoh, Y., Katayose, Y., Nakamura, S., Honkura, R., Nishimiya, S., Ueno, K., Mochizuki, A., Tanimoto, H., Tsugawa, H., Otsuki, Y., Ohashi, Y. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37:49-59 (1996)    Non-Patent Document 11: Murashige, T., Skoog, F. A revised medium for rapid proliferation and bio assays with tobacco tissue cultures. Physiol Plant 15(3), 473-497 (1962)